Cells and groups of cells are three-dimensional objects containing rich spatial information. Many different analytical probes, which preferentially bind to specific target receptors within a cell, are available to identify the distribution of different bio-molecules within a cell. There is a growing interest in studying cells as dynamic structures including numerous interacting feedback control systems. Understanding cells as dynamic structures, as opposed to static structures, can lead to the development of new drugs, better diagnostic procedures, more effective therapies, and better health care management strategies. However, achieving this improved understanding will likely require the ability to extract a far greater amount of information from cells than can currently be achieved using existing tools.
The principal technologies for cellular analysis are automated microscopy and flow cytometry. The information generated by these mature technologies, while useful, is often not as detailed as would be desired. Automated microscopy enables two-dimensional (2D) imaging of from one to three colors of cells on slides. Typical video frame rates limit kinetic studies to time intervals of 30 ms. Instruments known as flow cytometers currently provide vital information for clinical medicine and biomedical research by performing optical measurements on cells in liquid suspension. Whole blood, fractionated components of blood, suspensions of cells from biopsy specimens and from cell cultures, and suspensions of proteins and nucleic acid chains are some of the candidates suitable for analysis by flow cytometry.
In flow cytometers that are specifically designed for routine blood sample analysis, cell type classification is performed by measuring the angular distribution of light scattered by the cells, and the absorption of light by specially treated and stained cells. The approximate numbers of red blood cells, white blood cells of several types, and platelets are reported as the differential blood count. Some blood-related disorders can be detected as shifts in optical characteristics, as compared to baseline optical characteristics, and these shifts are indicative of morphological and histochemical cell abnormalities. Flow cytometers have been adapted for use with fluorescent antibody probes, which attach themselves to specific protein targets, and for use with fluorescent nucleic acid probes, which bind to specific DNA and RNA base sequences by hybridization. Such probes find application in the medical field for the detection and categorization of leukemia, as well as application in the fields of biomedical research and drug discovery. By employing these prior art techniques, flow cytometry can measure four to ten colors from living cells. However, prior art flow cytometry offers little spatial resolution, and no ability to study a cell over time. There is clearly a motivation to address the limitations of cell analysis technologies by providing an imaging platform that is configured for high speed and high sensitivity cell imaging.
A key issue that arises in cell analysis carried out with imaging systems is the measurement of the velocity of a cell or other object through the imaging system. Thus, many such systems include velocity detection components. A particularly useful technology for measuring object velocity in fluids is based on the insertion of a grating with alternating opaque and transparent parallel bars in the light path of a photo-sensor. Light from moving objects is modulated by the optical grating pattern to create a signal with a frequency directly proportional to the component of velocity perpendicular to the optical grating bars. If object motion is constrained to this perpendicular direction, then the frequency is equal to the true velocity divided by the period, or pitch, of the optical grating. A laser velocimeter based on this principle for measuring the velocity of a reflective surface moving relative to the instrument is disclosed in U.S. Pat. No. 3,432,237, issued on Mar. 11, 1969, and entitled “VELOCITY MEASURING DEVICE.” In the disclosed system of this patent, the target surface is illuminated with a continuous wave laser, and light scattered by the moving surface is collected by a lens and then delivered to a photosensitive detector through a grating. The bars of the optical grating are oriented perpendicular to the axis of motion. An electronic frequency measuring circuit is used to determine the frequency of the photosensitive detector. The frequency is conveyed directly to a display device for viewing and is converted to velocity.
An application of this method to objects suspended in fluid is disclosed in U.S. Pat. No. 3,953,126, issued on Apr. 27, 1976, and entitled “OPTICAL CONVOLUTION VELOCIMETER.” In the disclosed apparatus of this patent, light collimated by a lens passes through the flow of fluid and is reflected by a mirror with alternating bars of reflective and absorptive material. The reflective bars return light through the flow of fluid for collection by the lens. The lens focuses the reflected light on a photosensitive detector. An electronic circuit is used to estimate the frequency of the detector signal and to deliver the frequency to a display device for viewing as a velocity.
It should be noted that the hardware signal processors used in early implementations of laser velocimeters have largely been displaced by computation-based digital signal processors. The demands on the photosensor signal processors vary with the nature of the application, but the most stringent applications demand high speed and high accuracy, under conditions of low SNR and rapidly varying flow velocity.
An example of an effective method for extracting velocity from the photosensor signal of a grating-based laser velocimeter is disclosed in U.S. Pat. No. 5,859,694, issued on Jan. 12, 1999, and entitled “OPTICAL VELOCIMETER PROBE.” In this patent, the digitized photosensor signal is captured in blocks of samples for processing. For each block, the signal processor executes the steps of generating a complex signal using the Hilbert transform, auto-correlating the complex signal, and extracting the phase for each time sample of the auto-correlogram. The autocorrelation is performed using the steps of applying a complex Fourier transformation, squaring the magnitude of the spectrum, and then applying an inverse Fourier transformation. Finally, an optimization routine finds a best-fit velocity value for the phase samples. The method described in this patent has the advantage of building SNR and delivering accurate velocity estimates, given long signal segments. However, the method is computation intensive, limiting the rate at which the velocity estimate is updated using readily available processors.
Particularly useful implementations for modulating light from moving objects to measure their velocity, by inserting a periodic grating into the detector path, are described in commonly owned and assigned U.S. Pat. Nos. 6,532,061 and 6,507,391, both entitled “Measuring the velocity of small moving objects such as cells.” Flow imaging systems described in these two patents are configured to enable hydrodynamic focusing of a sample fluid within a flow cell. Light from objects entrained in a fluid passing through the flow cell is modulated by an optical grating to enable an indication of a velocity of the objects to be determined. That indication of velocity is employed to synchronize a time delay integration (TDI) detector to the flow of the fluid (and of the objects entrained therein).
It is significant that the ability of a flow imaging systems to accurately determine velocity, such as by using an optical grating as disclosed in the above-noted references, is only one of the abilities that should be provided by a preferred flow imaging system for analyzing cells. The ability of such a flow imaging system to automatically ensure that objects in flow remain properly focused is also highly desirable. To further enhance the utility of flow imaging systems, such as those implementing velocity detection based on modulating light with an optical grating, it would therefore be desirable to provide a method and apparatus for implementing an auto focus ability in such flow imaging systems. Optimized and predefined focal settings in flow imaging systems can be degraded by shock, movement of optical elements over the life of the system, temperature expansion coefficients of optical elements and related components, and temporal frequency changes occurring within a hydrodynamically focused fluidic core.